Complete sequence of Cynanchum rostellatum (Apocynaceae: Asclepiadoideae) chloroplast genome and its phylogenetic analysis

Abstract Cynanchum rostellatum (Turcz.) Liede and Khanum 2016 is a perennial herbaceous twining vine that is widely distributed in Japan, South Korea, the United States of America, and China. In this study, the complete chloroplast (cp) genome of C. rostellatum was sequenced using the Illumina platform and assembled for the first time. This plastome has a circular structure with a length of 160,641 bp. The GC content of the plastome was 37.82%. The cp genome contained 113 unique genes, including 79 protein-coding, 30 transfer RNA, and four ribosomal RNA genes. Phylogenetic analysis based on the complete cp genome sequences of the Asclepiadoideae subfamily showed that C. rostellatum was closely related to C. bungei in the genus Cynanchum. These results provide useful information for both phylogenetic research and the utilization of C. rostellatum.

Cynanchum rostellatum (Turcz.) Liede and Khanum 2016 is a perennial herbaceous twining vine of the Asclepiadoideae subfamily of Apocynaceae, with the heterotypic synonym Metaplexis japonica in the Flora Reipublicae Popularis Sinicae database (http://www.iplant.cn/; Ma and Clemants 2006). The herb contains milky latex and is widely distributed in Japan, South Korea, the United States of America, and China (Welsh and Anderson 1962). The fiber of C. rostellatum has considerable industrial application prospects, and the fruit, root, stem, leaf, seed hair, and milky juice of C. rostellatum have a long history in traditional Chinese medicine (Yang et al. 2006;Wang et al. 2020). The chemical components of C. rostellatum have been shown to have a variety of pharmacological activities, such as antitumor, antioxidant, antibacterial, immunosuppressive, and neuroprotective activities (Zhang et al. 2014;Wei et al. 2019). Although C. rostellatum has significant economic and medicinal value, genetic and evolutionary research is extremely rare. In this study, we report the complete chloroplast (cp) genome of C. rostellatum and examine its phylogenetic position within the subfamily Asclepiadoideae. It is expected to lay the foundation for further molecular studies and the utilization of this species.
Fresh leaves of C. rostellatum were collected from Xixia County, Nanyang City, Henan Province (33 38 0 N, 111 43 0 E). The specimen and DNA were deposited at the Herbarium of Henan University of Traditional Chinese Medicine, Henan, China (contact person: Lixin Pei, xlpxlp@aliyun.com), under voucher number HNPS2020-12-032. Total genomic DNA was extracted using a Dneasy Plant Mini Kit (Qiagen, CA, USA) according to the manufacturer's instructions (Cui et al. 2020). The genomic library for 150 bp paired-end sequencing was produced using the Illumina HiSeq 1500 platform (Illumina Inc., USA). The cp genome of C. auriculatum (NC029460) was used as a reference for assembly and annotation. The complete chloroplast genome was assembled using Getorganelle (v. 1.1.1; Jin et al. 2020), and annotated by Plann (Huang and Cronk 2015). The novel cp genome was submitted to the NCBI database (www.ncbi.nlm.nih.gov) under the GenBank accession number OL689165.
The cp genome of C. rostellatum contained circular double-stranded DNA and displayed a typical quadripartite structure, including two inverted repeat (IR) regions with lengths of 23,841 bp and 23,836 bp, separated by a large single-copy (LSC) region of 92,051 bp and a small single copy (SSC) region of 20,913 bp. The GC content of the plastome was 37.82%. The genome contained a set of 132 genes, of which 113 were unique and 19 were duplicated. A total of 87 protein-coding genes (79 unique genes) were annotated and were mainly involved in processes related to photosynthesis and gene expression. Eleven protein-coding genes (rps16, atpF, rpoC1, petB, petD, rpl16, two rpl2 genes, two ndhB genes, and ndhA) and eight tRNA genes (trnK-UUU, trnG-UCC, trnL-UAA, trnV-UAC, trnI-GAU, trnA-UGC, trnA-UGC, and trnI-GAU) contained one intron, and two genes (clpP and ycf3) contained two introns.
To elucidate the evolutionary relationship of C. rostellatum, the cp genomes of 21 Asclepiadoideae species were downloaded from the NCBI GenBank database. We aligned the plastomes using MAFFT and constructed a maximum likelihood (ML) tree ( Figure 1) using RAxML (v.8.2.9), using the GTRGAMMA model with 1000 rapid bootstrap replicates (Cui et al. 2020). Our plastome phylogeny showed that C. rostellatum is most closely related to C. bungei in the genus Cynanchum. Cynanchum rostellatum and seven Cynanchum species were clustered into one branch indicating that C. rostellatum was likely placed in the Cynanchum genus, and not in Metaplexis, during taxonomic classification. In conclusion, the cp genome of C. rostellatum provides a theoretical basis for a better understanding of the evolutionary patterns and for improving its taxonomic classification.

Ethical approval
No permission was necessary in this study for the sample collection. Smilax moranensis is widely distributed in North China and is not listed as a national key protected plant.